Tetra-, penta-, and hexa-nor-lanostane triterpenes from the medicinal fungus Ganoderma australe

Abstract Chemical investigation on the medicinal fungus Ganoderma australe led to the identification of ten new nor-lanostane triterpenes, namely two hexa-nor ones, ganoaustratetraenones A (1) and B (2), five penta-nor ones, ganoaustraldehydes A–E (3–7), and three tetra-nor ones ganoaustrenoic acids A–C (8–10). The chemical structures along with the absolute configurations were determined by extensive spectroscopic analysis of 1D & 2D NMR and HRESIMS data. The postulated biosynthesis pathways of these compounds were proposed. Ganoaustraldehydes A (3) and B (4) showed moderate inhibition against nitric oxide production in RAW264.7 macrophage cells with the respective IC50 values of 32.5, 34.2 µM (the IC50 of positive control pyrrolidine dithiocarbamate was 20.0 µM). Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-022-00356-x.


Introduction
The genus Ganoderma contains more than 80 species of wood decaying fungi mostly distributed in tropical and subtropical areas. Different species of Ganoderma have been used in Traditional Chinese Medicine for thousands of years for the treatment of many kinds of diseases, for example, hypertension, respiratory diseases, gastrointestinal disorders, autoimmune diseases [1]. The two species, G. lucidum and G. sinense were recorded in the recent five editions of Chinese Pharmacopoeia with the name "Lingzhi" from the year 2000. Numerous studies have shown that polysaccharides and the main secondary metabolites-triterpenoids, are responsible for the biological activities of Ganoderma, such as immunoregulatory, antiviral, hepatoprotective effects [2][3][4][5][6][7]. Besides, meroterpenes (farnesyl hydroquinones), the other main constituent in Ganoderma, have attracted much attention in recent years [8,9]. Ganoderma triterpenes are always characterized by structural poly-oxygenations [6]. Among the reported structures, the popular positions been oxygenated are C-3, C-7, C-11, C-15, C-23, Page 2 of 10 Zhou et al. Natural Products and Bioprospecting (2022) 12:32 and C-26. The oxygenations in the side chain sometimes trigger the C-C bond cleavage by retro-aldol reaction or oxidative cleavage to yield nor-lanostane, a minor group of Ganoderema triterpenes, such as C24 (hexa-nor), C27 (tri-nor), and C-25 (penta-nor) lanostanes [5,6]. Ganoderma australe is regarded as an alternative of the official-recognized species G. lucidum, and is used as a folk medicine in some ethnic minority areas of Yunnan Province, China. This fungus, however, was chemically under-investigated compared to other easily widely used Ganoderma species, such as G. lucidum, G. cochlear, and G. sinense. Previous studies on the secondary metabolites of G. australe have led to the isolation of some lanostane triterpenes [10][11][12][13], meroterpenoids [14,15], and alkaloids [14]. In this study, we would like to report ten new nor-lanostane triterpenes isolated from G. australe, of which the structures are assigned by extensive NMR and HRESIMS spectroscopic analysis. The new structures are classified into tetra-nor-, pentanor-, and hexa-nor-lanostanes, and are featured by C-6 oxygenation or α,β-unsaturated aldehyde groups, which are unusual modifications in the reported Ganoderma triterpenes, thereby inspiring a biosynthetic proposal of these compounds.
The stereochemistry of the C-17-C-21 double bond was assigned as Z configuration according to the ROESY cross peaks between H-21 (δ H 10.03) and H-12 (δ H 3.08) (Fig. 3). The OH-6 was determined to be α configuration by the diagnostic ROESY signals of H-6/H 3 -19 (δ H 1.25) (Fig. 3). Therefore, compound 4 was elucidated as shown in Fig. 1. The molecular formula of compounds 5 and 6 ( Fig. 1) were same with those of 3 and 4, respectively. The 1D NMR data of 5 and 6 (Tables 1, 3 and 2) also exhibited high similarity to those of 3 and 4, respectively, thus indicating that 5 and 6 were the respective structure congeners of 3 and 4. Analysis of the 2D NMR spectra of 5 and 6 enabled us to identify the only difference between these two pairs of compounds, which was the configuration of C-17-C-21 double bond (Fig. 2). The diagnostic ROESY correlations of H 3 -21(δ H 1.80)/H-12 (δ H 3.05, 3.10), and H-22 (δ H 10.00)/H-16 (δ H 3.12 in 5; 3.19 in 6) (Fig. 3) suggested the E configuration of C-17-C-21 double bonds of 5 and 6 (Fig. 3). The configuration of OH-6 of 6 was determined as α orientation by the ROESY correlation of H-6/H 3 -19 (Fig. 3). Therefore, compounds 5 and 6 were identified as ganoaustraldehydes C and D, respectively.
Since these nor-lanostane are characterized by unusual C-6 oxygenation, and α,β-aldehyde groups, a biosynthetic proposal is postulated as shown in Scheme 1.

The anti-NO production activity of the isolates
All the isolates were subjected to screening their inhibition against the NO production in murine monocytic RAW 264.7 macrophages. As a result, only compounds 3 and 4 displayed moderate inhibitory activities with IC 50 values of 32.5, 34.2 µM, respectively.

Conclusions
Ten previously undescribed nor-lanostane triterpenes were isolated from the fruiting bodies of Ganoderma australe. The chemical structures of the compounds were determined with the aid of extensive NMR and HRESIMS spectroscopic analysis. These compounds are featured by tetra-, penta-, and hexa-nor-lanostane scaffold, and by unusual oxidative modifications at C-6. These findings put the diversity of nor-lanostanes of Ganoderma origin one step forward.

Fungal material
The fruiting bodies of Ganoderma australe were collected in Tongbiguan Natural Reserve, Dehong, Yunnan Province, China, in 2016, and identified by Prof. Yu-Cheng Dai, who is a mushroom research in Institute of Microbiology, Beijing Forestry University. A voucher specimen of G. australe was deposited in the Mushroom Bioactive Natural Products Research Group at South-Central Minzu University.